Compositions and process relating to macrocytic anemias



United States Patent 0 MAR91TE ANER AS Lul aby, 2794 Webb Ave, Blew Yorh 68, NY.

dune in, 1959, Ser. No. 820,596 12 {CL 16765) No Brewing.

The invention relates to a method and to a composition for the alleviation and prevention of a metabolic condition incident to the development of an anemia.

More particularly it pertains to the alleviation of macrocytic anemias and includes correlated improvements and discoveries whereby those conditions are wholly or to a marked extent alleviated.

An object of the invention is to provide an efiective method for the treatment of metabolic conditions which may lead to the development of anemias and particularly megaloblastic and macrocytic anemias.

A further object of the invention is the provision of a method which readily corrects certain types of macrocytic anemias.

A particular object of the invention is the provision of a method for the alleviation and prevention of megaloblastic and macrocytic anemias which entails the adinist'ration of histidine in a composition suitable for such administration.

A further object is to provide histidine-containin com positions suitable for prevention and alleviation of megaloblastic macrocytic anemias.

A still further object of the invention is the provision of a method for efiective diagnosis and determination of folic acid deficiency.

Another object of the invention is the provision of a method for differentiation of megaloblastic anemias due to folic acid deficiency from those anemias due to vitamin B deficiency.

Additional objects of the invention are the provision of a method for effective diagnosis and determination of liver disease characterized by impairment of folic acid and histidine metabolism, and for efiective diagnosis and determination of toxemia of pregnancy where the folic acid and histidine metabohsm are impaired.

(Ether objects or" the invention will in part be obvious and will in part appear hereinafter.

The invention accordingly comprises the several steps and the relation of one or more of such steps with respect to each of the others and the product possessing the features, properties, and the relation of constituents, which are exemplified in the following detailed procedure, and the scope of the invention will be indicated in the claims.

Practice of the invention provides a method and a composition for the treatment, i.e., alleviation of metabolic conditions incident to the development of anemias especially macrocytic a emias which entails the administration of the amino a histidine to individuals having metabolic conditions which may lead to an anemia and especially to those individuals afiiicted with certain types of macrocytic anemias. Y I have found that such administration gives an unexpected and unique result, namely, the anemia is promptly and materially improved. Further, I have found that the administration of histidine is elfective in certain conditions, notably pregnancy, and in certain diseases particularly liver disease which are l-movm to lead to macrocytic anemia, for it delays or prevents the onset of anemia, especially megaloblastic macrocytic anemia in these conditions and diseases.

Practice of the therapeutic aspect of this invention is .also effective for the mleviation of conditions arising from a body deficiency or loss of histidine, such situations occurring in, among other, pregnancy. Megaloblastic macrocytic anemia has been known to be due to, among other things, a specific deficiency or lack of availability to the body cells, of either folic acid, vitamin B or a combination of both. The histidine administration is particularly effective in correcting the megaloblastic macrocytic anemia in those instances where there is an actual or a metabolic deficiency of folic acid. The unexpected and surprising aspect of this action is that the anemia is corrected even though the folic acid deficiency is not corrected.

The administration of histidine has a beneficial therapeutic effect in the following types of anemias, among others:

(1) Nutritional macrocytic anemia.

(2) Macrocytic anemia associated with sprue.

(3) Megaloblastic anemia associated with gastro-intestinal malabsorption syndromes or after intestinal shunting operations.

(4) Megaloblastic or macrocytic anemia of pregnancy.

(5) Megaloblastic anemia of infancy and early child hood.

(6) Macrocytic anemia associated with liver insufiiciency and cirrhosis.

(7) Macrocytic anemia associated with toxemia of pregnancy.

(8) Certain instances of pernicious anemia, namely,

those complicated by a simultaneous folic acid deficiency.

(9) Anemia due to histidine deficiency.

The method has been utilized viz., the administration of histidine, in several score of individuals with certain other types of anemias and diseases and I have found no similar effect. This includes anemia due to acute leukemia, anemia associated with certain types of cancer, Hodgkins disease, aplastic anemia, Cooleys anemia, sickle cell anemia, anemia of prematurity and those nutritional anemias not complicated by folic acid or histidine deficiency.

In the therapeutic use of histidine, the surprising and unexpected finding was that patients treated with histidine promptly began to feel better and their anemia quickly improved, even though the folic acid deficiency was not corrected as indicated by a striking increase in the urinary excretion of formiminoglutamic acid within about 48 to 72 hours from the onset of histidine therapy. Subsequently I found that those patients who would excrete large amounts of formiminoglutamic acid upon administration of adequate and appropriate amounts of histidine as set forth below are precisely the opens who responded best clinically and hemotologically to histidine dosages. l resumably, histidine is producing its effect in these individuals by a different (still unknown) metabolic pathway. it appears that those individuals whose body tissue folic acid content is low, sub-normal or borderline, require more than the amounts of histidine available to them from dietary sources, in order to maintain clinical, hematologic and physiologic normality. Therein resides the utility, uniqueness, unexpectedness, importance and value of the histidine method.

I have also found in recent studies that individuals with folic acid deficiency excrete abnormally large amounts of histidine in their urine. This uniary loss of histidine leads to histidine depletion in the body. In normal pregnancy it was found that greater than normal amounts of histidine were excreted in the urine commencing from about the sixth week of pregnancy. In pregnant women with megaloblastic macrocytic anemia, which usually occurs in the last trimester of pregnancy and which was not responsive to vitamin B adm nistration but responsive to small doses of folic acid, hence indicating 'folic acid deficiency, even larger amounts of histidine were found in the urine than in non-anemic pregnant women in the same period of gestation.

After folic acid administration to these. patients with megaloblastic macrocytic anemia who have abnormally increased urinary histidine excretion, the urinary histidine output decreases strikingly. Thus, this evidence indicates that in folic acid deficiency there is body depletion of histidine due to increased urinary loss of this amino acid in particular. The beneficial therapeutic effect of the administration of histidine in the amounts set forth herein in folic acid deficiency anemias is greater than can be achieved with the amounts of histidine available even in high protein diets 'which provide about 2 grams of histidine per day. High protein diet therapyis inadequate to achieve the beneficial effects contemplated by this invention wherein a dosage of at least 5 grams per day is employed. In such folic acid deficiency anemias treated with folic acid more rapid and beneficial therapeutic effects are obtained when at least the critical minimum of histidine is administered in combination with the administration of the folic acid.

I have also found, most unaccountably, that the therapeutic activity of histidine for these purposes is strikingly enhanced beyond any possible additive contribution by the addition to histidine of para-aminobenzoic acid. I have also discovered that similar strikingly enhanced effects are achieved by the addition to histidine of folic acid. The compositions are predominantly histidine with a minor proportion of the para aminobenzoic acid or folic acid. The para-aminobenzoic acid may be present a in an amount from about 1% to about 45% by weight of the histidine calculated as the free base. The folic acid may be present in an amount from about 0.0006% to about 0.25% by weight of histidine calculated as the free base. The proportion by weight of histidine to folic acid will be from about 165,000 to 1 to about 400 to 1. I have also found that the addition of serine (including the D, L, and DL forms and mixtures and salts thereof) to histidine similarly and unaccountably gives greater effect than with either histidine or serine alone. The serine may be present in an amount from about 6% to about 100% by weight of histidine calculated as the free base. benzoic acid, histidine with folic acid, or histidine with serine results in truly synergistic combinations wherein the observed clinical results is far greater than may possiblybe accounted for by similar dosages of either of the components or" these combinations.

Furthermore the histidine may be mixed with the folic acid, para-aminobenzoic acid and serine and the combination administered with beneficial effects.

In addition to para-arninobenzoic acid itself, its non- The combination of histidine with para-amino- Salts of the histidines or combinations of them are equally effective as an equivalent amount of the free base. Various salts involving any non-toxic anion or cation may be used. Examples are, the mono or di-hydrochloride sulphates and phosphates, etc. and such non-toxic a solid, or more particularly, a pharmaceutical carrier and for this purpose utilization may be made of lactose, a soluble starch and the like. Furthermore, the carrier may be a liquid preferably an aqueous medium, and

when the administration is subcutaneously, intramuscw' larly, or intravenously, the histidine would be in sterile water or physiological saline. I

In certain instances the administration of histidine or mixtures therewith of either folic acid, paraarniriobendissolved Zoic acid or serine as a dietary supplement may be ,de-'

sirable where prophylaxis is desired in subjects where anemic tendencies are suspected or anticipated.

In assessing the hematologic effectiveness of anti-.

anemia materials after administration oftest substances to anemic patients, the subsequent rise of the peripheral red blood cell count and the hemoglobinconcentration are followed, and the patterns of the rise and fall of viously known anti-anemic materials for 'megaloblastic anemia, begins to rise on the 4th to th day, reaches its peak on the 7th to 10th day, and then sharply decreases again, returning to almost baseline levels by the 14th to 21st day. With highly potent anti-anemic materials this response may be shifted toward a day earlier, with low potency substances, a day or so later. In these anemias, there often are low white blood cell and platelet counts,

which rise to normal values following efiective therapy.

toxic salts may be employed as for example alkali metal para-aminobenzoate, such as sodium-or potassium salts,

alkaline-earth metal para-aminobenzoate, as for example the calcium salt. Similarly non-toxic salts of serine, as for example the acidic salts such as the hydrochlorides, or the basic salts such as the sodium salts may be effectively employed. .7

The therapeutic method is carried out as follows: Histidine preferably L-histidine, although D-histidine, DL-

histidine, racemic and various'combinations thereof and salts thereof are administered by mouth as a powder, tablet or in solution.

may be required. Usually about 3.8 grams may be administered 3 times a day before meals for a total daily dose of about 11.5 grams. The dosage is continued until the desired hematological improvement is attained.

such dosage is administered, a method foretfective diagnosis of folic acid'deficiency, whether the folic acid deficiency is caused by an actual deficiency or by the inability of a human body to utilize folic acid in its metabolic processes as it involves in particular histidine metabolism. The new method now provides a" test for .folic acid deficiency that is specific, sensitive and reliable.

The invention provides a specific and effective test to determine in a person with macrocytic megaloblastic anemia the existence of folic acid deficiency and to diE ferentiate such anemia from that due to vitamin B deficiency. Such diagnostic procedure is most useful in the differentiation of anemias for the reason that the clinical manifestations of anemias responsive to treat ment with folic acid are for the most part otherwisev indistinguishable from those anemias which are responsive to treatment with vitamin B There is thus provided an effective diagnostic tool, the; results of which are'reliably indicative to the physician of the mode The reticulocyte count characteristically in response to pre-,

of treatment to be employed. Thus, the absence of a significantly elevated urinary formiminoglutamic acid level following administration of histidine or its nontoxic salts signifies the absence of folic acid deficiency and differentiates the megaloblastic anemia as due to vitamin B deficiency, while the presence of a significantly elevated formirninoglutamic acid level identifies a folic acid deficiency, although in the latter instance the possibility of an accompanying vitamin B deficiency is not ruled out.

Furthermore, the administration of the critical amount of histidine daily provides a spech'ic and effective test to determine existence of liver disease in which folic old and histidine metabolism are adversely afiected. The test identifies patients with liver disease when megaloblastic anemia and toxemia of pregnancy are not presout. if a patient has macr cytic megaloblastic anemia and the physicians' examination and labor tory tests indicate abse se of liver disease or toxemia of preg nancy, the results of the test indicate that there is a nutritional deficiency of folic acid, providing the patient has not received any folic acid metabolic antagonist.

Moreover, in pregnant individuals that test provides a method for diagnosis of toxemia of pregnancy, and a pos ive test for that condition when other tests are eq ivocal. Patients with toxemia of pregnancy may have high blood pressure and this differentiates pregnant individuals with high blood pressure (hypertension) due to other causes from those to toxemia.

in folic acid deficiency an imino acid metabolic intermediate, formiminoglutamic acid, accumulates, which apparentry arises from degradation of histidine. Formiminoglutamic acid is normally further degraded to glutamic acid in the presence of the physiologically active tom of folic acid, viz. tetrahydrofolic acid. In the absence of tetrahydrofolic acid (either because of a dietary deficiency of the folic acid or failure of gastrointestinal absorption, r failure of conversion to tetrahydrofolic acid, due to the lack of necessary liver enzymes, or the pressure of a folic acid antagonist, or inasmuch as increased metabolic requirements reduce the stores) formiminoglutamic acid accumulates in the human body and is excreted in the urine. Excretion of this compound is an indicator of metabolic or physiologic folic acid deficiency. However, many individuals with anemia and wi h what should be a complicating clinical or subclinical folic acid deficiency were found who did not excrete formiminoglutamic acid. The test for r'ormimino glutarnic acid in the urine thus appeared to be unsatisfactory for diagnosis. However, I have found that if the subject of diagnosis is given :1 'dine over a period of at least about 48 hours in the critical amount of about 5 to about grams per day of histidine calculated as the free base the assay of formiminoglutamic acid in the urine becomes an effective and reliable test for distinguishing folic acid responsive anemias from other anemias not so responsive.

Dosages at the lower level of the critical range are employed with infants and children.

Furthermore, after the administration of histidine for a period of at least about 48 hours, the formiminoglu tamic acid assay becomes an effective and reliable test for diagnosis and determination of liver disease characterized by impairment of folic acid and histidine meta olism, and for diagnosis and determination of toxemia of pregnancy where the folic acid and histidine rretabolism are afiected.

The diagnostic method is carried out as follows: a metabolic load of L-histidine in a dosage of about 5 grams daily to about 15 grams daily calculated as the free base is administered to a human host for at least 48 hours and preferably for about 47 to about 72 hours. The histidine employed may be administered in the forms described above for therapeutic administration. During the last 24 hour period and preferably at the end of the period a sample of urine is collected under concentrated acid to maintain the pH of the urine below 2.0. Quantitative measurement is made of the urinary formimino glutamic acid concentration. Effective quantitative measurement is made by a technique that is based upon enzymatic reactions involved in the metabolism of folic acid and formirninoglutamic acid. The assay method employed involves converting formiminoglutamic acid in the urine to 5,10-methenyl tetrahydrofolic acid by enzymatic and chemical conversion and the concentration of the latter is determined by spectrophotometric measurement of its optical density at its Wave lengths of maximum absorption such being either 350 m 365 m c, or 380 mu.

he quantitative measurement may be particularly ad vantageously carried out as follows:

The enzyme reagent used is prepared as fo lows:

Dried defatted liver powder is obtained by homogeuiz ing 100 grams of fresh pork liver with 500 ml. of acetone in a Waring Biendor for one minute. The mixture is filterer. through a Buchner funnel and the liver cakes rehomogenizcd with 500 ml. acetone for one minute and filtered. The cake is broken up and allowed to dry in air. 106 grams of the dried liver powder are stirred With 1000 ml. water for 15 minutes at room temperature. The suspension is centrifuged at 20,000X gravity for 15 minutes and the precipitate discarded. To 1,000 ml. of supernatant solution add 214 grams ammonium sulfate and keep at l3 C. for one hour. The precipitate formed is collected by centrifuging at 20,0()0 gravity for 15 minutes at l3 C. The precipitate is suspended in 30 ml. of water and stored in the refrigerator overnight. The latter suspension is then centrifuged for two hours at 30,0GOX gravity at l-3 C. for two hours, and the supernatant discarded. The precipitate is dissolved in 30 ml. of 6.2 M sodium acetate. After two hours, it is cen trifuged and the precipitate is discarded. The remaining solution contains both formiminoglutamic acid transferase and cyclodeaminase. it is kept frozen at C. until used.

The tetrahydrofolic acid reagent used is prepared by the following new method which is simpler and safer than the methods heretofore employed.

1% mg. platinum oxide (Adams catalyst) is suspended in ml. glacial acetic acid. Hydrogen gas is bubbled into the solution at atmospheric pressure until platinum black precipitates. 200 mg. l-folic acid suspended in 25 ml. glacial acetic acid is then added to the above solu tion. Hydrogen is again bubbled into this reaction mix ture at atmospheric pressure until reaction is completed (4-6 hours). Completion of reaction can be recognized when solution no longer absorbs hydrogen gas as deter mined by a water manometer. When reaction is completed, the excess hydrogen is Washed out by bubbling helium gas through the reaction mixture, followed by nitrogen gas to keep oxygen out of the atmosphere in the reaction vessel above the solution. Entire reaction mixt re is then poured into stoppered centrifuge tubes, nitrogen again bubbled through, tubes sealed tightly and centrifuged at 1930-2090 rpm. for 10 to 20 minutes. The clear supernatant containing the dl-tetrahydrofolic acid is decanted into screw top test tubes and sealed.

By employing the above steps the yield of tetrahydro folic acid is significantly greater than by methods not employing nitrogen gas and by separation of the supernatant containing the tetrahydrofolic acid from the pre cipitate by filtration in the open atmosphere. This can be stored at 20 C. for 6 to 12 months. To use, 25 ml. are removed into a suction flask and connected to vacuum at room temperature until glacial acetic acid is evaporated. To each residue equivalent to 25 ml. of original solution, 51.1 ml. of 1.1 molar mercaptoethanol are added. Concentrated 16 N KOH is added dropwise to the latter solution, stirring until all the tetrahydrofolic acid goes into solution and pH reaches 7.0-7.5. Yields of to of theoretical maximum are usually ob l the total volume of 1.0 ml.

To 0.1 ml. of a test urine sample, pI-l adjusted to ,be-

tween 6 and 7, placed in a' 3.0 ml. test tube, is added 0.25 ml. of the enzyme reagent, 0.1 ml. of the tetrahydrofolic acid reagent and 0.1 ml. 1 M potassium phosphate bufier of pH 7.2. Water is then added to bring Ir" the concentration of formirninoglutamic acid in urine is expected to be low, additional arnounts of pH 6 to 7 urine can be added as seems appropriate, instead of Water, to bring the final volume to 1.0 ml. If the urine concentration of formiminoglutamic acid is expected to be high, the urine is diluted appropriately 1:10, 1:100, 1:500 or as necessary, before use.

The mixture is allowed to react at 25 C. for 30 minutes. 0.3 ml. 10% perchloric acid was then added'and the mixture allowed to react at room temperature for 2 hours and then centrifuged at about 1500 r.p.m. for 10 minutes.

The supernatant is removed into a 1.5 ml. silica cuvette with an 0.5 or 1.0 cm. light path and its optical density is read at 350 millimicrons (111,14). A urine control for the non specific absorption of the mixture of urine, tetrahydrofolic acid and I phosphate buffer reagents is prepared in an identical fashion as set forth above for the test urine, but substituting 0.25 ml. water for the 0.25 ml. of enzyme reagent, and the optical density at 350 my. of this sample is also obtained. An enzyme control for the non-specific absorption of theenzyme preparation in combination with the tetrahydrofolic acid and phosphate buffer is also prepared in an identical manner as set forth for the test urine, but a 0.1 ml. of water was substituted for the'0.1 ml. of urine. The optical density of this sample at 350 mg is also obtained. The optical density reading of the urine and enzyme control samples are added and then subtracted from the optical density reading of the test urine sample. The latter result, the corrected optical density, is then multiplied by a factor relating optical density units to micrograms of formirninoglutamic acid per m1. of solution. The latter factor is obtained by .substitutingOl ml. of a solution containing 17.4 micro? grams of pure (synthetic) formirninoglutamic acid for the 0.1 ml. of test urine, and'determining the optical density at 350 m of the resulting reaction mixture ob tained in a similar manner.-

The optical density'can also be read at 365 my. or 380- m Where slightly lesser extinction maxima of 5,10

'methenyl tetrahydrofolic acid also exist.

It has been found that in non-folic acid deficient individuals subjected to the above-described histidine metabohc loading, the urinary formiminoglutamic acid con centration does not exceed about 30 to about 35 1 /1111. In sharp contrast to this, individuals withmegaloblastic anemia due to folic acid deficiency, under identical c0ndition's, exhibit urinary formiminoglutamic acid concentrations of from 3 to 1000 times greater than do indivviduals not having folic acid deficiency. Under the described diagnostic procedure, no patient with uncompli-' cated Addisonian pernicious anemia in relapse, recognized as being due to a deficiency of vitamin B showed formirninoglutamic acid excretion in excess of about 35 macrocytic anemias:

total dose of 15 grams.

the histidine the reticulocyte percentage in the blood had risen to 6.0 and by the fifth day reached a peak of 10.2. This is an unusually prompt response and is quicker than 1 l 'y/ml. All patients with other types of megaloblastic anemia showed an excretion value sufficiently above normal to befunequivocal. The concentrationsvaried from about -2000 y/ml. Certain individuals iuwhom fohc acid deficiency could at best be classified as borderlinlf or sub-clinical were distinctly diagnosable.

to Addisonian pernicious anemia could not be induced to excrete urinay formirninoglutamic acid above 35 'y/ml.

toxemia of pregnancy are comparable to theresults ob tained in folic acid deficient individuals. It has been found that patients who did not have liver disease and who did not have toxemia of pregnancy when subjected to the above described :histidine loading had urinary formiminogluta'mic acid excretions thatdid not exceed about 30 to about 35 /ml. In sharp contrast, under identical conditions patients with liver diseaseand those with toxemia of pregnancy exhibited urinary formiminoglutamic acid concentrations of from 3100 times as much.

As illustrative embodiments of the invention the following examples are presented to illustrate the therapeutic effectiveness of histidine, in various megaloblastic EXAMPLE 1 L-histidine, as the mono-hydrochloride salt dissolved in an appropriate aqueous fluid, e.g., tomato juice, was orally administered to apatient diagnosed as having sprue with megaloblastic macrocytic anemia. Five grams were given three times daily before meals for one day for a On the second day after taking observed with other previously known anti-megaloblastic anemia medications such as folic acid, vitamin B 9 or liver extract. The red blood cell count rose from 2.03 to 2.85 per cu. mm. and the hemoglobin concentration from 8.0 to 11.0 grams percent in the same five day period, also an unusually prompt and materially striking effect. These results indicate that the histidine treatment is acting more directly or supplying the necessary missing ingredient for hemoglobin and red cell formation more efiiciently in such patients than the previously used standard treatments such vasfolic acid, vitamin B liver extract, or

high protein diet. Bone marrow examination done on the sixth day after start of therapy showed distinctive improvement of the megaloblastic features, thus further demonstrating the curative nature of the histidine therapy. In addition, the patient improved clinically as evidenced by being stronger, more alert, better appetite, and decrease in size and quantity'of the stools. The day after administration of the histidine, the patient volunteered that he felt subjectively improved' Summary of Blood Findings in Example I EXAMPLE 2 L-histidine monohydrochloride was orally administered 'to a patient diagnosed as having nutritional macrocytic anemia in three divided does of 5 grams each day in a flavored aqueous menstruum, for a total does of 15 grams for only day only. By the 7 post treatment day, the

Normal individuals as well as those with megaloblastic anemia due reticulocytes rose to 4.0 percent, the white cells to 5,350 per cu. mm., the red count to 2.4 million/mm. and the hemoglobin to 10.6 grams percent. The patient felt stronger and more alert. Repeated bone marrow examination on 5 post treatment day showed improvement. This illustrates an excellent response to a low total dose of the medication in a patient who is not very anemic.

Two Weeks later, the patient partially relapsed. The red count fell to 2.17 million/mmfi, the hemoglobin to 9.0 grams percent, the White cell count to 4,000 and reticulocytes 0.2 percent. At this time, the patient was given 30 milligrams of folic acid orally daily for an eleven day period. By the 9 day following this treatment, the reticulocytes had risen to a peal: of 10.0 percent, the red count to 2.76 million per mm. and the hemoglobin to 11.0 grams percent. On the 14 after iolic acid, the red cell count was only 2.72, the hemoglobin l0.8 grams percent. This illustrates that in the not very anemic tient, even large doses of previously used treatment such as folic acid, does not produce a very dramatic response. By comparison, the previous course with histidine produced an excellent response.

Summary of Blood Findings in Example 2 EXAMPLE 3 L-histidine monohydrochloride was orally administered to a patient diagnosed as having megaloblastie macrocytic anemia of pregnancy in a flavored aqueous menstruum, 5 grams 4 times daily for a total daily dose or" 20 grams, for a seven day period. The hemoglobin and red cell count began to rise on the 2nd post treatment day. The hemoglobin reached a level of 12 grams percent and the red cell count 3.8 millions/mm. on the 6th post treatment day. This is again a quicker and more dramatic increase of these blood values and a pr npter alleviation of the anemia than by previously kl'lCnI medications such as folic acid. Concomitantly with the improvement of the blood, the patient volunteered she felt stronger. The patient excreted considerable quantities of tormimlnoglutamic acid in her urine begining on the third post medication day. This finding plus her previously insignificant blood respon e to vitamin B and liver extract therapy is consistent With the presence of a folic acid deliciency in the patient.

Summary Blood F iizdz'itgs in Example 3 Before After Blood count of patient showed Histiaine Histidine Treatment Treatment For 7 Days Red blood cells (millions/mmfi) 2 4 3. 8 Hemoglobin (grams percent) 9 12. 0 Reticulocytes (maximum petcent) 0 3 4. O

Summary of Blood Findings in Example 4 EXAMPLE 5 L-histidine monohydrochloride was orally administered to a patient diagnosed as having megaloblastic macrocytic anemia with liver insufiiciency, 5 grams in an aqueous nienstruurn, 3 times daily for a total daily dose of grams. Treatment was continued for days. The hemoglobin red cell count began to rise promptly. The retlculocytes rose promptly and followed a slightly extended pattern for typical therapeutically efiective materials, reaching 13.5% on the 5th day. This patient also excreted large amounts of formirnino-glutamic acid in the urine alter the histidine administration was begun. The patient improved clinically with the histidine treatment.

This illustrates that his-tidine administration can produce a complete remission and alleviation of certain .egioblastic anemias, particularly in those instances in which the histidine administration is followed in 48 to 72 hours by increased urinary excretion of iormirninoglutamic acid.

Summary of Blood Findings in Example 5 Before After Blood count of patient showed Histidine Histidine Treatment Treatment For 30 Days Red blood cells (millions/mm. 1. 4 4. 0 Hemoglobin (gr ms percent) 6.0 13. 8 Reticulocytes (maximum percent) 5. 0 1 13. 5

1 (5th day peak.)

XAMPLE The potentiating cli'ect of small doses of folic acid given simultaneously with histidine is illustrated by the followi g example of the r use and effect in a patient with n itionai rnegaloblastic macrocytic anemia and gastrointestinal malabsorption syndrome.

Below are listed the increase in the blood hemoglobin, red blood cell count and the reticulocyte percentage which occurred in this patient following three consecutive therapeutic eriods of five days in which the following were given: irst period: l-folic acid, 0.9 millidaily was given by mouth in three equally divided doses of 0.3 mflligram in a flavored liquid carrier, /2 hour before meals. Second period: L-lnstidine monolaydrochloride Was given in three doses of 5 grams each in a flavored liquid carrier, /2 hour before meals for a total daily dose of 15 grams. Third period: l-folic acid, 0.3 milligram Was given in combination with 5 grams of L- histidine monohydrochloride, for 3 doses daily in a fiavored liquid carrier /2 hour before meals.

Compositions in Treatment Increase after each period of Histi- Folio Histidine Acid dine Plus Folic Acid Hemoglobin (grams percent) a 1.0 1. 5 3. 2 Red blood cells (millions/mind) 0. 7 0.6 1 8 Reticulocytes (max. percent) 6. 2 5.1 22 3 EXAMPLE 7 The enhancing therapeutic efiect of p-aminobenzoic in a patient diagnosed as having megaloblastic macrocytic anema due to the malabsorption syndrome associated'with sprue.

After L-histidine monohydrochloride was administered orallyina flavored aqueous carrier in 3 divided 4 gram doses for a total daily dose of 12 grams for six days the increases in the patients hemoglobin red blood cells and maximum reticulocyte percentage are shown below in column 1. At a subsequent six day period, 1 gram of p-aminobenzoic acid was administered three times daily in a flavored aqueous carrier. In a third six day treatment period, 1 gram of para-aminobenzoic acid and 4- grams L-histidine monohydrochloride in combination which provided a total daily dose of L-histidine monohydrochloride ofl2 grams and p-aminobenzoic acid of 3 grams were administered together in a flavored aqueous carrier. The increases in the hemoglobin, red blood cell, count and reticulocytes of the patients blood are shown below in columns 2 and 3 respectively for the'second and third treatment period. l l

The efiects following administration of p-aminobenzoic acid plus histidine was greater than could be expected from either substance alone.

EXAMPLE 8 The potentiating therapeutic efiect of serine when administered with histidine is illustrated by the following example of their use and eiiect in a patient with megaloblastic macrocytic anemia of pregnancy.

In column 1 below is shown the increase of the hemoglobin, red blood cell count and maximum reticulocyte percentage'following 15 grams daily of L-histidine monohydrochloride orally administered in 3 equally divided doses in an aqueous carrier. In column 2 below are the results following 10 grams daily of DL serine orally administered in a iiavored aqueous carrier in 3 equally divided doses. in column 3 the results following oral administration of the combination of 5 grams of L-histiine monohydrochloride and 3.3 grams of DL serine three times daily in an aqueous carrier for a total daily dose 7 of. grams of the histidine. salt and 10 grams of the DL serine.

Compositions in Treatment Increase after each period ol (1) (2) (3) Histi- Histi- Serine dine dine Plus Serine Hemoglobin (grams percent). 2.0 0. 4 3. 2 Red Blood Cells (millions/mm?) 0. 3 O. 1 1.0 Beticulocytes (max. percent) 2. 5 3.0 4. 5

As illustrated by the examples above, but not limited to these specific types, histidine treatment as set forth produces prompt and distinct beneficial hematological and clinical eflects in the conditions and anemias claimed herein. In these situations it produces an increase of blood hemoglobin quicker and to a greater extent and a more rapid reticuiocyte response, indicating more direct eiiectiveness than previously known medications such as folic acid, vitamin B liver extract or high protein diet alone. It appears that its hematologic effect is greatest upon hemoglobin formation, less upon red cell'produo ticn, although it will effect an increase in all the blood cellular elements, i.e., the white cells, platelets and red cells.

I-listidine would thus appear to be or supply a necessary ingredient for blood regeneration in the conditions 7 and disorders outlined herein. One can only hypothesize at this time how it produces these effects. It is particularly effective in'folic acid deficiency states. ltrrnay be that there is an inverse metabolic relationship between folic acid and histidine or its products. When one is low, more of the other is needed. It may be that histidine in ordin'aryamounts is metabolically unavailable or the body becomes histidine deficient through urinary loss in folic acid deficiency states but is hematologically effective when the amount employed in the therapeutic aspect of the invention is administered. When present in such larger amounts it may short circuit the folic acid metabolic pathway in some still unknown manner. It is thus a significant improvement on folic acid and the other therapeutics. It is an important ingredient for the treatment and prevention of the conditions and disorders set forth. Herein lies not only the uniqueness of histidine administration, but also itsadditional utility. it may be given as a preventive dietary supplement in those physiologic or metabolic conditions which may give rise to or result in macrocytic anemias, in particular megaloblastic anemia.

Histidine when given with folic acid potentiates the effect i tained.

illustrative embodiments of the application and per formance of the diagnostic method are as follows:

EXAMPLE 9 A patient was clinically demonstrated to have nutritional megaloblastic macrocytic anemia by blood, bone marrow and other tests. A metabolic load of L-histidine monohydrochloride, 15 grams daily, was administered to l the patient in'three divided doses one-half hour before meals in a flavored aqueous carrier for 48 hours. Urine excreted by'the patient during the final 24 hour period of administration was collected under concentrated acid and the urinary formiminoglutamic acid output during that period was measured by the enzyme reaction technique set forth above. The concentration of formiminoglutamic acid in a, urine sample collected at the end of the 48 hour period was also measured. The calculated amount of forniiminoglutamic acid found was 98 ug/ml.

or" urine in the sample taken atjthe end of the 48 hour.

period, and 191.1v mg. in the totalvolume of urine excreted during the 24-48 hour period. At the end of 72 hours of continued histidine loading the urinary formiminoglutarnic'acid values were even greater.

EXAMPLE 10 r was 7.6 ,ug/ml. and the total output during the urine concentration of formiminog utarnio acid e final 24 hours was 6.0 mg.

Examples 9 and 10 illustrate how increased urinary formiminoglutamic acid concentration and total 24 hour urinary output of formiminoglutamic acid after the histidine metabolic load test set forth identifies a patient with folic acid deficienc and differentiates such a subiect with megaloblastic macrocytic rmemia from a subject with an identical type of anemia but which is caused by a body deficiency of vitamin B incident to Addisonian pernicious anemia in relapse.

EXAMPLE 11 To a patient diagnosed as having toxemia of pregnancy grams of L-bistidine monohydrochloride per day were orally administered in three divided doses one-half hour before meals in an aqueous carrier for a 48 hour period, and the urine collected and measured. A sample of the patients urine at the end of the 48 hour period indicated a formiminoglutamic acid concentration of 215.5 ,ug./ml.; the final 24 hour output of formiminoglutamic acid was 249 milligrams.

Tlsu's illustrates that in a patient with toxemia of pregnancy and no overt evidence of megaloblastic anemia increased urinary formiminoglutamic acid excretion occurs. in other pregnant patients without megaloblastic anemia or preceding liver disease, where other evidence for toxemia was equivocal, the histidine metabolic loading test unequivocally indicated the existence of toxemia of pregnancy.

EXAMPLE 12 A 15 grams per d y loading of L-histidine monohydrochloride was orally administered in three divided doses one-half hour before meals in an aqueous carrier to a patient diagnosed as having hypochromic anemia of pregnancy for a 48-hour period and the urine collected and measured. A sample of the patients urine at the end of the 48 hour period had a formim ioglutamic acid concentration of 6.1 ,rg/ml. and the final 24 hour output of formim'moglutamic acid was 3.6 mg.

This example illustrates that in pregnancy associated with non-megaloblastic anemia, the histidine metabolic load test does not result in increased urinary formiminoglutamic acid excretion, indicating no deficiency of folio acid, or interference with folic acid or histidine metabolism as occurs in toxemia of pregnancy.

EXAMPLE l3 L-histidine monohydrochloride was administered to a child diagnosed as having liver disease due to biliary cirrhosis in 3 divided doses of 3 grams each an aqueous carrier over a 48 hour period and the urine collected and measured for formiminoglutamic acid. A sample of the patients urine obtained at the end of the 48 hour load test period had a formiminoglutamic acid concentration of 405 The patients urine collected during the immediately preceding 24 hour period had a total formiminoglutarnic acid output of 190 milligrams. This illus trates that the occurrence of increased formhninoglutamic acid in the urine after histidine metabolic loading in an individual without megaloblastic macrocytio anemia or toxemia of pregnancy is diagnostic of liver disease.

the course of my development of this histidine treatment I have administered histidine monohydrocmoride in as large a daily dose as 85 grams without ill effect on a patient, and have administered it in various smaller daily dosages for periods up to six months and have found no toxic, harmful or deleterious efiect in the patient or any interference with other nutrients or medications.

It will thus be seen that the objects sets forth above, among those made apparent from the preceding description, are efiiciently attained, and, since certain changes and certain modifications in the composition which emid body the invention may be made in carrying out the above method without departing from the scope of the invention, it is intended that all matter contained in the above descrip on shall be interpreted as illustrative and not in a lin ng sense.

It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention here n described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.

This application is a continuation-impart of application erial No. 784,098, fled December 31, 1958, now aban- 0 .ed.

I claim:

1. A method for the alleviation of megaloblastic macrocytic anemias which comprises administering to a human host aiiiicted with such an anemia a member of the group consisting of histidine and the non-toxic salts thereof in a dosage of from about 5 to about 33 grams, calculated as free base, daily.

2. A method for the alleviation of megaloblastic macrocytic anemias which comprises administering to a human host affiicted with such an anemia a member of the group consisting of histidine and the non-toxic salts thereof together with folic acid, in a daily dosage from about 5 to about 33 grams of histidine calculated as free base, the amount of folic acid being from about 0.0006% to about 0.25% by weight of the histidine calculated as free base. 1

3. A method for the alleviation of megaloblastic macrocytic anemias which comprises administering to a human host afflicted with such an anemia 21 member of the group consisting of histidine and the non-toxic salts thereof together with a member of the group consisting of paraaminobenz'oic acid and the non-toxic salts thereof, in a daily dosage from about 5 to about 33 grams of histidine calculated as free base, the amount of member of the group consisting of para-aminobenzoic acid and the non-toxic salts thereof being from about 1% to about 45% by weight of the histidine calculated as free base.

4. A method for the alleviation of megaloblastic macrocytic anemias which comprises administering to a uman host afllicted with such an anemia a member of the group consisting of histidine and the non-toxic salts thereof together with a member of the group consisting of serine and the non-toxic salts thereof, in a daily dosage from about 5 to about 33' grams of lustidine calculated as free base, the amount of the member of the group consisting of serine and the non-toxic salts thereof being from about 6% to about by weight of the histidine calculated as free base.

5. A method of enhancing the therapeutic efiect of histidine and the non-toxic salts thereof adm nistered in the treatment of megaloblastic macrocytic anemias which comprises administering concurrently therewith folic acid in an amount corresponding to between about 0.0006% and about 0.25% by weight of the administered histidine calculated as free base.

6. A method of enhancing the therapeutic eifect of histidine and the non-toxic salts thereof administered in the treatment of megaloblastic macrocytic anemias which comprises administering concurrently therewith a member of the group consisting of para-aminobenzoic acid and the nontoxic salts thereof in an amount corresponding to between about 1% to about 45% by weight of the administered histidine calculated as a free base.

7. A method of enhancing the therapeutic eifect of histidine and the non-toxic salts thereof administered in the treatment of megaloblastic macrocytic anemias which comprises administering concurrently therewith a member of the group consisting of serine and the non-toxic salts thereof in an amount corresponding to between about 6% and about 100% by weight of the administered histidine calculated as free base.

8. A synergistic composition of matter for the alleviation of megaloblastic macrocytic anemias comprising a member of the group consisting of histidine and the nontoxic salts thereof in combination with folic acid and a pharmaceutical carrier, the daily dosage range of said member being between about ,5 and about 33 grams,

calculated as free base, the amount of folic acid being between about 0.0006% and about 0.25% by weight of the histidine calculated as free base;

9. A synergistic composition of matter for the alleviation of megaloblastic macrocytic anemias comprising a first member selected from the group consisting of histidineand the non-toxic salts thereof, a second member selected from the group consisting of para-aminobenzoic acid and the non-toxic salts thereof and a pharmaceutical carrier, the dosage range of said first member being between about and about 33 grams, calculated as free base,

daily and the amount of said second member being between about 1% and about 45% by weight or" the histidine calculated as free base.

10. A synergistic composition of matter for the alleviation of megaloblastic macrocytic anerrnas comprising a first member selected from the group consisting of histidine and the non-toxic salts thereof, a second member selected from the group consisting of serine and the nontoxic salts thereof and a pharmaceutical carrier, the dosage range of said first member being between about 5 and about 33 grams, calculated as free base, daily and the amount of said second member being between about 6% and about 100% by weight of the histidine calculated as free base;

11. A method for the diagnosis of folic acid deficiency which comprises administering to a human host a member of the group consisting of histidine and the non-toxic salts thereof in a daily dosage of from about 5 to about 15 grams calculated as free base for a period of from about 48 to about 72 hours, and at substantially the termination of said period determining the urinary formiminoglutamic acid excretion.

12. The method of diagnosing folic acid deficiency which comprises determining the formiminoglutamic acid content of the urine of a human host to Whom a member of the group consistingof histidine and the non-toxic salts thereof is'administered in a daily dosage of'between about 5 and about 15 grams, calculated as free base, aformiminoglutamic acid level in excess of about 30-35 micrograms per ml. identifying folic acid deficiency, a formiminoglutarnic acid level below about 30-35 micrograms per ml. at least 48 hours after initial administration of said member'signifying absence of folic acid deficiency.- V

References Cited in the-file of this patent-- UNITED STATES PATENTS 1,625,165 Seaton Apr. 19, 1927 2,457,820 Howe Jan. 4, 1 949 2,531,097 Alpert Nov. 21, 1950 2,791,533 Segal May 7, 1957 OTHER REFERENCES Physician Bulletin, June 1947, pp. 77-81.

Lederle: The Common Forms of Anemia, January 1947, pp. 13-18. V 9 Williams et al.: Chem. Abs., vol. 49, 1955, 7016d. Sebrell et al.: The Vitamins, vol. Ill, 1954, p. 39, p. 55,

p. 59, Academic Press. 7

Fontes et a1.: Compt. rend. Soc. Biol, vol; 106, pp. f 592-4, 1931.

Specter: Handbook of Biological Data, 1956, p. 238.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No; 3,157,575 November 17 1964 Adrian Leonard Luhby It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected belo Column 2 line 51 for "opens" read ones column 3 line 47 for "results" read result --g column 4 line 26 for "paraaminoben-" read para-aminobencolumn 5, line 23 for "that" read this line 40, for "pressure" read presence same column 5 line 72 for 47 read 48 column 7 line l4 for "of" first occurrence read to column 8, line 74 for "does" read dose line 75 for "only", first occurrence read one column 9 line l7 for "2.72" read 282 (SEAL) Signed and sealed this 30th day of March 1965 lltlest:

ERNEST W. SWIDER' EDWARD J. BRENNER Attesting Officer Commissioner of Patents 

1. A METHOD FOR THE ALLEVIATION OF MEGALOBLASTIC MACROCYTIC ANEMIAS WHICH COMPRISES ADMINISTERING TO A HUMAN HOST AFFICTED WITH SUCH AN ANEMIA A MEMBER OF THE GROUP CONSISTING OF HISTIDINE AND THE NON-TOXIC SALTS THEREOF IN A DOSAGE OF FROM ABOUT 5 TO ABOUT 33 GRAMS, CALCULATED AS FREE BASE, DAILY. 